Preparation and characterization of a potent, long-lasting recombinant human serum albumin-interferon-α2b fusion protein expressed in Pichia pastoris

Abstract

A long-lasting recombinant human serum albumin-interferon-α2b fusion protein (rHSA/IFNα2b) was prepared and its structure and biological activities were studied. rHSA/IFNα2b was expressed in methylotrophic yeast Pichia pastoris with HSA’s natural signal peptide and purified by dye affinity chromatography, hydrophobic interaction chromatography, ion exchange chromatography and Sephadex G25. Purity of the prepared rHSA/IFNα2b was greater than 97% analyzed by non-reduced SDS–PAGE and RP-HPLC. Structure and biological activities of the prepared rHSA/IFNα2b were characterized by physical, chemical and biological methods. Its p I was 5.3 and showed a single band on IEF gel. Molecular weight determined by MALDI-TOF was 86004.3 ± 29.2. Amino-terminal and carboxyl-terminal amino acid sequences were identical to predicted sequence. Its specific activity in vitro was 6.3 ± 0.8 × 10 5 IU/mg fusion protein, retaining about 1.4% of that of unmodified rIFNα on a molar basis. After administered in monkeys, significant increases of 2′,5′-oligoadenylate synthetase activity relative to IFN-α were maintained for 14 days in serum and the rHSA/IFNα2b showed more potent biological activity than IFN-α on a molar basis. Therefore, markedly improved in vivo biological activity of rHSA/IFNα2b could exhibit more potent antiviral activity than IFNα2b in future clinical trials.